DESCRIPTION: The Human Immunodeficiency Virus Type 1 (HIV-1), the major causative agent of Acquired Immune Deficiency Syndrome (AIDS), encodes the accessory protein Nef. Nef plays a crucial role in primate lentivirus replication and pathogenesis. Nef also enhances HIV-1 infectivity in single-cycle infection assays and accelerates HIV-1 replication in vitro. The focus of this application is to elucidate the molecular mechanism by which Nef enhances HIV-1 infection. Based on our two key discoveries that Nef is associated with the HIV-1 core and that targeting HIV-1 infection to an endocytic entry pathway relieves the requirement for Nef, a mechanistic model is proposed in which Nef facilitates the intracellular transport of the viral genome. In this model, Nef tethers the incoming viral nucleoprotein complex to the endocytic machinery via simultaneous interactions with the viral core and with cellular adaptor proteins, resulting in routing of the incoming core to an intracellular compartment permissive for reverse transcription. Three specific predictions of this model will be tested. First, the hypothesis that Nef is active in the virion will be tested using two novel technologies for delivery of proteins into virions. The second hypothesis, that interaction of Nef with the core is required for infectivity enhancement, will be tested through identification of the target of Nef binding and genetic analysis of the interaction. The third hypothesis, that interaction of Nef with cellular adaptor proteins is required for efficient HIV-1 infection, will be tested through mutational analysis of Nef binding to cellular adaptor proteins and assays of HIV-1 infection. Finally, the relationship between the effect of Nef in single cycle infection assays and in continuous replication assays will be determined by analyzing the kinetics of wild type and Nef-defective HIV-1 replication in T cell cultures inoculated with either HIV-1 particles or with known numbers of infected cells. These experiments will reveal novel functional targets of Nef in HIV-1 infection, will contribute to our mechanistic understanding of Nef function, and will define the relative requirements for Nef in cell-free virus infection vs. cell-to-cell transmission. These studies should therefore facilitate the development of a biochemical assay for rapid identification of specific inhibitors of Nef as potential antiviral drugs.